Site-specific recombination system to manipulate the plastid genome of higher plants

ABSTRACT

A site-specific recombination system and methods of use thereof are disclosed for manipulating the plastid genome of higher plants.

This application is a §371 filing of PCT/US00/25930 filed Sep. 21, 2000which in turn claims priority to U.S. provisional application 60/155,007and 60/211,139 filed Sep. 21, 1999 and Jun. 13, 2000, respectively, eachof which is incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to the fields of transgenic plants and molecularbiology. More specifically, DNA constructs and methods of use thereofare provided which facilitate the excision of target DNA sequences fromtransplastomic plants.

BACKGROUND OF THE INVENTION

Several publications are referenced in this application by author nameand year of publication in parentheses in order to more fully describethe state of the art to which this invention pertains. Full citationsfor these reference can be found at the end of the specification. Thedisclosure of each of these publications is incorporated by referenceherein.

The plastid genetic system of higher plants is highly polyploid. Forexample, in a tobacco leaf there are as many as 100 chloroplasts, eachcarrying ˜100 identical genome copies, a total of 10,000 copies in aleaf cell. High-level protein expression, lack of pollen transmissionand the feasibility to engineer polycistronic expression units make theplastid genome an attractive alternative to nuclear engineering. Plastidtransformation vectors often contain a selective marker, most commonly aspectinomycin resistance (aadA) gene, flanked by plastid DNA sequencestargeting insertion of the marker gene by homologous recombination intothe plastid gnome. Genes of commercial value but lacking a selectablephenotype are physically linked to the selective marker and the twogenes are integrated together as a block of heterologous sequences.Plastid transformation is accomplished by biolistic DNA delivery orpolyethylene glycol induced uptake of the transforming DNA followed byselection for the antibiotic resistance marker to ensure preferentialpropagation of plastids with transformed genome copies. As the result,all the 10,000 wild-type plastid genome copies in a cell are replacedwith transgenic copies during a gradual process (Maliga, 1993).

Incorporation of a selectable marker gene is essential to ensurepreferential maintenance of the transformed plastid genome copies.However, once transformation is accomplished, maintenance of the markergene is undesirable. One problem may be the metabolic burden imposed bythe expression of the selectable marker gene. For example FLARE-S, theproduct of the marker gene with good prospects to transform cerealchloroplasts, accumulates up to 18% of the total soluble cellularprotein (Khan and Maliga 1999). The second problem is the relativelyhigh potential for horizontal transfer of plastid marker genes tomicrobes (Tepfer 1989; Dröge et al. 1998; Sylvanen 1999), as commonlyused plastid maker gene constructs are efficiently expressed in E. coli(Carrer et al. 1993; Svab and Maliga 1993). Therefore, having plastidmarker genes in commercial products is undesirable.

SUMMARY OF THE INVENTION

In accordance with the present invention, methods and systems areprovided which facilitate the manipulation of the plastid genomes ofhigher plants. The methods and systems of the invention may be employedto remove heterologous sequences from the plastid genome, such asselectable marker genes following successful isolation of transformedprogeny. Alternatively, they may be designed to remove endogenous genesinvolved in plant cell metabolism, growth, development and fertility.

In one embodiment of the invention, a site specific recombination methodfor removal of predetermined nucleic acid sequences from the plastidgenome is provided. The method comprises providing a first nucleic acidconstruct, the construct comprising a promoter being operably linked toa nucleic acid encoding an optional plastid targeting transit sequencewhich is in turn operably linked to a nucleic acid encoding a proteinhaving excision activity, the construct further comprising a firstselectable marker encoding nucleic acid having plant specific 5′ and 3′regulatory nucleic acid sequences. The method also entails the use of asecond DNA construct, the second construct comprising an secondselectable marker encoding nucleic acid and excision sites. The secondconstruct optionally contains a gene of interest and further comprisesflanking plastid targeting nucleic acid sequences which facilitatehomologous recombination into said plastid genome. The second DNAconstruct is introduced into plant cell and the cells are cultured inthe presence of a selection agent, thereby selecting for those plantcells expressing the proteins encoded by said second DNA construct. Thefirst DNA construct is then introduced into cells having the secondconstruct in the presence of a selection agent and those plant cellsexpressing proteins encoded by said first construct are selected. Ifpresent, the excising activity acts on the excision sites, therebyexcising said predetermined target sequence. Plants may then beregenerated from plant cells obtained by the foregoing method.

Proteins having excision activity suitable for the practice of theinvention include, without limitation, CRE, flippase, resolvase, FLP,SSV1-encoded integrase, and transposase. Sequences corresponding toexcision sites suitable for the practice of the invention, include, forexample, LOX sequences, and frt sequences.

A variety of selection of agents may be selected. These include withoutlimitation, kanamycin, gentamycin, spectinomycin, streptomycin andhygromycin, phosphinotricin, basta, glyphosate and bromoxynil.

In an alternative embodiment, a site specific recombination method forremoval of predetermined nucleic acid sequences from the plastid genomeis provided. The method comprising providing a first nucleic acidconstruct, said construct comprising a regulated promoter being operablylinked to a nucleic acid encoding an optional plastid targeting transitsequence which is operably linked to a nucleic acid encoding a proteinhaving excision activity, said construct optionally further comprising afirst selectable marker encoding nucleic acid having plant specific 5′and 3′ regulatory nucleic acid sequences. A second DNA construct is alsoprovided, said second construct comprising an second selectable markerencoding nucleic acid and excision sites, said second construct furthercomprising flanking plastid targeting nucleic acid sequences whichfacilitate homologous recombination into said plastid genome at apredetermined target sequence such that excision sites flank saidpredetermined target sequence following homologous recombination andintroducing said second DNA construct into a plant cell. The plant cellso generated is then cultured in the presence of a selection agent,thereby selecting for those plant cells expressing the proteins encodedby said second DNA construct. A plant is then regenerated from cellscontaining the second construct and the first DNA construct isintroduced into these cells in the presence of a selection agent andthose plant cells expressing proteins encoded by said first constructare selected. The excising activity then acts on the excision sites,thereby excising said predetermined target sequence.

Regulatable promoters suitable for this embodiment of the inventioninclude, without limitation, inducible promoters, tissue specificpromoters, developmentally regulated promoters and chemically induciblepromoters.

Candidate predetermined target sequences, may include for example genesassociated with male sterility, clpP, ribosomal proteins, ribosomaloperon sequences.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram depicting CRE-mediated excision andintegration of DNA segments.

FIG. 2 is a map of a plastid transformation vector pSAC48, with codabracketed by direct loxP sites. Positions of plastid genes rrn16, trnV,rps12/7 (Shinozaki et al. 1986), the aadA and codA transgenes andrelevant restriction sites are marked.

FIG. 3 is a map of an Agrobacterium binary vector pPZP212 with aplastid-targeted Ssu-tp-cre gene. Marked are: Agrobacterium Left andRight Border fragments; the kanamycin resistance (neo) gene; P2′promoter; SSU transit peptide (ssu-tp); cre coding region; recognitionsequences for restriction enzymes BamHI, EcoRI, HindIII, NcoI, NheI andXbaI.

FIG. 4 shows maps of the plastid genome >codA> deletion derivatives.Shown are the plastid targeting region of vector pSAC48; the map of sameregion of the wild-type plastid genome (Nt-wt); the map of the plastidgenome with CRE-mediated deletion of coda via the lox sites; and the mapof the plastid genome with deletion via Prrn sequences lacking trnV,aadA and coda. Positions of plastid genes rrn16, trnV and rps12/7(Shinozaki et al. 1986), aadA and codA transgenes, primers (O1–O4) andrelevant restriction sites (AI, ApaI; EV, EcoRV) are marked.

FIG. 5 is a gel showing PCR amplification which confirms CRE-mediateddeletion of codA from the plastid genome. Primers O1 and O2 (FIG. 3)amplified the 0.7-kb fragment of the deleted region. Same primersamplify the 2.0-kb aadA-codA fragment in tester lines Nt-pSAC48-21A andNt-pSAC-16C (no transgenic Cre gene). No specific fragment was obtainedin wild-type DNA sample and in Cre1-10 line. The lines obtained arelisted in Table 1.

FIG. 6 shows the results of DNA gel blot analysis wherein plastid genomestructure was determined in the indicated plant samples. Total cellularDNA was isolated from the leaves of plants listed in Table 1 anddigested with the ApaI and EcoRV restriction endonucleases. The probeswere the wild-type ApaI-EcoRV plastid targeting region and the aadA(NcoI-XbaI fragment) and coda (NcoI-XbaI fragment) coding regions. Thehybridizing fragments are marked in FIG. 3.

FIG. 7 are gels showing uniformity of plastid genome populations in theSsu-tp-cre transformed plants. Total cellular DNA extracted from severalleaves was probed with the ApaI-EcoRV targeting region probe. Numbersidentify leaves from which DNA was extracted. For example, sevendifferent leaves were probed from the Cre1-3 plant. For details, seeBrief Description of FIG. 6.

FIGS. 8A and 8B are gels of PCR analysis confirming CRE-mediateddeletion of coda in seedlings obtained by pollination with Ssu-tp-creactivator lines. 5-day old seedlings were tested from the crossNt-pSAC48-21A as maternal parent and Cre2-200 and Cre2-300 activatorlines as pollen parents. Amplification products are also shown forcontrols Nt-pSAC48-21A selfed seedling (48 self), wild-type (wt), theparental plant (48P) and the Cre1-3 plant. FIG. 8A: The coda region wasamplified with the O1/O2 primers: the size of aadA-codA fragment is 2.0kb; the coda deletion fragment is 0.7 kb (FIG. 4). FIG. 8B: Testing forcre sequences by PCR amplification with the Cre1/Cre3 oligonucleotides.

FIG. 9 is a diagram of the plastid transformation pSAC38 with the >neo<bracketed by inverted lox sites. Positions of plastid genes rrn16, trnVand rps12/7 (Shinozaki et al., 1986), the aadA and codA transgenes andrelevant restriction sites are marked.

FIG. 10 shows a map of the plastid genome containing the >neo< inversionconstruct. Shown are the plastid targeting region of vector pSAC38; themap of the same region of the wild-type plastid genome (Nt-wt); map ofthe plastid genome with CRE-mediated inversion of neo via the lox sites.Positions of the plastid genes rrn16, trnV and rps12/7 (Shinozaki etal., 1986) aadA and neo transgenes, primers (O1–O4) and relevantrestriction sites (BamHI) are marked.

FIG. 11 shows the results of DNA gel blot analysis for the determinationof plastid genome structure of CRE-activated >neo< plants by DNA gelblot analysis. Total cellular DNA was digested with the BamHIrestriction endonuclease. The probes was the wild-type ApaI-EcoRVplastid targeting region. The hybridizing fragments are marked in FIG.10.

FIG. 12 shows an exemplary monocistronic inversion vector. The gene ofinterest (goi) coding region is flanked by inverted lox sites(triangles). CRE activates goi expression by inversion, so that thecoding strand is transcribed. rrn16, trnV and rps12/7 are plastid genes(Shinozaki et al. 1986).

FIG. 13 shows an alternative dicistronic lox inversion vector. Note thatthe inverted lox sites flank the selective marker (aadA) and goi, andonly one gene is expressed. rrn16, trnV and rps12/7 are plastid genes(Shinozaki et al. 1986).

FIG. 14 shows a basic tobacco plastid lox deletion vector. The vectorprovides is a suitable backbone for vector construction and targetsinsertions into the trnV-rps12/7 intergenic region.

FIG. 15 shows a tobacco plastid lox >aadA> deletion vector. rrn16, trnVand rps12/7 are plastid genes (Shinozaki et al. 1986).

FIG. 16 shows a tobacco constitutive >aadA> goi dicistronic deletionvector. rrn16, trnV and rps12/7 are plastid genes and are described in(Shinozaki et al. 1986).

FIG. 17 shows a tobacco constitutive goi >aadA> dicistronic deletionvector. Note that vectors shown in FIG. 16 and FIG. 17 differ in therelative order of marker gene and the gene of interest. rrn16, trnV andrps12/7 are plastid genes (Shinozaki et al. 1986).

FIG. 18 shows a tobacco constitutive goi >aadA> dicistronic deletionvector, in which expression of aadA is dependent on translationalcoupling. Note that in this construct only one leader sequence isutilized. rrn16, trnV and rps12/7 are plastid genes (Shinozaki et al.1986).

FIG. 19 shows a tobacco inducible lox deletion vector. Expression of goiis dependent on aadA excision. rrn16, trnV and rps12/7 are plastid genes(Shinozaki et al. 1986). Abbreviations: P, promoter; T, 3′ untranslatedregion; L1 is 5′ leader sequence.

FIG. 20 shows a vector suitable for Cre-mediated deletion of clpP genefrom the plastid genome. The region of engineered plastid genome shownis the sequence contained in the plastid transformation vector. The clpPExons are dark boxes, the Introns are open boxes. Map position ofplastid genes psbB, rps12 Exon I and rp120 is also shown.

DETAILED DESCRIPTION OF THE INVENTION

The following definitions are provided to aid in understanding thesubject matter regarded as the invention.

Heteroplastomic refers to the presence of a mixed population ofdifferent plastid genomes within a single plastid or in a population ofplastids contained in plant cells or tissues.

Homoplastomic refers to a pure population of plastid genomes, eitherwithin a plastid or within a population contained in plant cells andtissues. Homoplastomic plastids, cells or tissues are genetically stablebecause they contain only one type of plastid genome. Hence, they remainhomoplastomic even after the selection pressure has been removed, andselfed progeny are also homoplastomic. For purposes of the presentinvention, heteroplastomic populations of genomes that are functionallyhomoplastomic (i.e., contain only minor populations of wild-type DNA ortransformed genomes with sequence variations) may be referred to hereinas “functionally homoplastomic” or “substantially homoplastomic.” Thesetypes of cells or tissues can be readily purified to a homoplastomicstate by continued selection.

Plastome refers to the genome of a plastid.

Transplastome refers to a transformed plastid genome.

Transformation of plastids refers to the stable integration oftransforming DNA into the plastid genome that is transmitted to the seedprogeny of plants containing the transformed plastids.

Selectable marker gene refers to a gene that upon expression confers aphenotype by which successfully transformed plastids or cells or tissuescarrying the transformed plastid can be identified.

Transforming DNA refers to homologous DNA, or heterologous DNA flankedby homologous DNA, which when introduced into plastids becomes part ofthe plastid genome by homologous recombination.

Operably linked refers to two different regions or two separate genesspliced together in a construct such that both regions will function topromote gene expression and/or protein translation.

“Nucleic acid” or a “nucleic acid molecule” as used herein refers to anyDNA or RNA molecule, either single or double stranded and, if singlestranded, the molecule of its complementary sequence in either linear orcircular form. In discussing nucleic acid molecules, a sequence orstructure of a particular nucleic acid molecule may be described hereinaccording to the normal convention of providing the sequence in the 5′to 3′ direction. With reference to nucleic acids of the invention, theterm “isolated nucleic acid” is sometimes used. This term, when appliedto DNA, refers to a DNA molecule that is separated from sequences withwhich it is immediately contiguous in the naturally occurring genome ofthe organism in which it originated. For example, an “isolated nucleicacid” may comprise a DNA molecule inserted into a vector, such as aplasmid or virus vector, or integrated into the genomic DNA of aprokaryotic or eukaryotic cell or host organism.

When applied to RNA, the term “isolated nucleic acid” refers primarilyto an RNA molecule encoded by an isolated DNA molecule as defined above.Alternatively, the term may refer to an RNA molecule that has beensufficiently separated from other nucleic acids with which it would beassociated in its natural state (i.e., in cells or tissues). An isolatednucleic acid (either DNA or RNA) may further represent a moleculeproduced directly by biological or synthetic means and separated fromother components present during its production.

The terms “percent similarity”, “percent identity” and “percenthomology” when referring to a particular sequence are used as set forthin the University of Wisconsin GCG software program.

The term “functional” as used herein implies that the nucleic or aminoacid sequence is functional for the recited assay or purpose.

The phrase “consisting essentially of” when referring to a particularnucleotide or amino acid means a sequence having the properties of agiven SEQ ID No. For example, when used in reference to an amino acidsequence, the phrase includes the sequence per se and molecularmodifications that would not affect the basic and novel characteristicsof the sequence.

A “replicon” is any genetic element, for example, a plasmid, cosmid;bacmid, phage or virus, that is capable of replication largely under itsown control. A replicon may be either RNA or DNA and may be single ordouble stranded.

A “vector” is a replicon, such as a plasmid, cosmid, bacmid, phage orvirus, to which another genetic sequence or element (either DNA or RNA)may be attached so as to bring about the replication of the attachedsequence or element.

An “expression operon” refers to a nucleic acid segment that may possesstranscriptional and translational control sequences, such as promoters,enhancers, translational start signals (e.g., ATG or AUG codons),polyadenylation signals, terminators, and the like, and which facilitatethe expression of a polypeptide coding sequence in a host cell ororganism.

The term “oligonucleotide,” as used herein refers to primers and probesof the present invention, and is defined as a nucleic acid moleculecomprised of two or more ribo- or deoxyribonucleotides, preferably morethan three. The exact size of the oligonucleotide will depend on variousfactors and on the particular application and use of theoligonucleotide.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15–25 or morenucleotides, although it may contain fewer nucleotides. The probesherein are selected to be “substantially” complementary to differentstrands of a particular target nucleic acid sequence. This means thatthe probes must be sufficiently complementary so as to be able to“specifically hybridize” or anneal with their respective target strandsunder a set of pre-determined conditions. Therefore, the probe sequenceneed not reflect the exact complementary sequence of the target. Forexample, a non-complementary nucleotide fragment may be attached to the5′ or 3′ end of the probe, with the remainder of the probe sequencebeing complementary to the target strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the sequence of the target nucleic acid to anneal therewithspecifically.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single-stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3′terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield an primer extension product. The primer mayvary in length depending on the particular conditions and requirement ofthe application. For example, in diagnostic applications, theoligonucleotide primer is typically 15–25 or more nucleotides in length.The primer must be of sufficient complementarity to the desired templateto prime the synthesis of the desired extension product, that is, to beable anneal with the desired template strand in a manner sufficient toprovide the 3′ hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template. For example, anon-complementary nucleotide sequence may be attached to the 5′ end ofan otherwise complementary primer. Alternatively, non-complementarybases may be interspersed within the oligonucleotide primer sequence,provided that the primer sequence has sufficient complementarity withthe sequence of the desired template strand to functionally provide atemplate-primer complex for the synthesis of the extension product.Amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form may besubstituted for any L-amino acid residue, provided the desiredproperties of the polypeptide are retained.

All amino-acid residue sequences represented herein conform to theconventional left-to-right amino-terminus to carboxy-terminusorientation.

The term “tag,” “tag sequence” or “protein tag” refers to a chemicalmoiety, either a nucleotide, oligonucleotide, polynucleotide or an aminoacid, peptide or protein or other chemical, that when added to anothersequence, provides additional utility or confers useful properties,particularly in the detection or isolation, to that sequence. Thus, forexample, a homopolymer nucleic acid sequence or a nucleic acid sequencecomplementary to a capture oligonucleotide may be added to a primer orprobe sequence to facilitate the subsequent isolation of an extensionproduct or hybridized product. In the case of protein tags, histidineresidues (e.g., 4 to 8 consecutive histidine residues) may be added toeither the amino- or carboxy-terminus of a protein to facilitate proteinisolation by chelating metal chromatography. Alternatively, amino acidsequences, peptides, proteins or fusion partners representing epitopesor binding determinants reactive with specific antibody molecules orother molecules (e.g., flag epitope, c-myc epitope, transmembraneepitope of the influenza A virus hemaglutinin protein, protein A,cellulose binding domain, calmodulin binding protein, maltose bindingprotein, chitin binding domain, glutathione S-transferase, and the like)may be added to proteins to facilitate protein isolation by proceduressuch as affinity or immunoaffinity chromatography. Chemical tag moietiesinclude such molecules as biotin, which may be added to either nucleicacids or proteins and facilitates isolation or detection by interactionwith avidin reagents, and the like. Numerous other tag moieties areknown to, and can be envisioned by, the trained artisan, and arecontemplated to be within the scope of this definition.

As used herein, the terms “reporter,” “reporter system”, “reportergene,” or “reporter gene product” shall mean an operative genetic systemin which a nucleic acid comprises a gene that encodes a product thatwhen expressed produces a reporter signal that is a readily measurable,e.g., by biological assay, immunoassay, radioimmunoassay, or bycalorimetric, fluorogenic, chemiluminescent or other methods. Thenucleic acid may be either RNA or DNA, linear or circular, single ordouble stranded, antisense or sense polarity, and is operatively linkedto the necessary control elements for the expression of the reportergene product. The required control elements will vary according to thenature of the reporter system and whether the reporter gene is in theform of DNA or RNA, but may include, but not be limited to, suchelements as promoters, enhancers, translational control sequences, polyA addition signals, transcriptional termination signals and the like.

The terms “transform”, “transfect”, “transduce”, shall refer to anymethod or means by which a nucleic acid is introduced into a cell orhost organism and may be used interchangeably to convey the samemeaning. Such methods include, but are not limited to, transfection,electroporation, microinjection, PEG-fusion, biolistic bombardment andthe like.

A “clone” or “clonal cell population” is a population of cells derivedfrom a single cell or common ancestor by mitosis.

A “cell line” is a clone of a primary cell or cell population that iscapable of stable growth in vitro for many generations.

Cre-Mediated Site Specific Recombination

The plastid genome of higher plants is present in 100–10,000 copies percell. Incorporation of a selectable marker gene is essential to ensurepreferential maintenance of the transformed plastid genome copiescarrying useful genes with no selectable phenotype. However, oncetransformation is accomplished, maintenance of the marker gene isundesirable. In accordance with the present invention, a bacteriophagePlCRE-loxP site-specific recombination system is provided which issuitable for efficient elimination of marker genes from the plastidgenome. The system exemplified herein has two components: a plastidtester strain carrying a cytosine deaminase (coda) transgene flanked bylox sites conferring sensitivity to 5-fluorocytosine and a nuclear CREline carrying a nuclear-encoded, plastid-targeted CRE. Both the plastidtester (no CRE activity) and the nuclear CRE line (no lox sequence) weregenetically stable. However, coda was eliminated at a very fast ratewhen the plastid-targeted CRE was introduced into the plastid testerstrain by transformation or crossing. The gene for the nuclear-encodedCRE was subsequently separated from the transformed plastids bysegregation in the seed progeny. Excision of coda by CRE was oftenaccompanied by deletion of a plastid genome segment flanked by shortdirectly repeated sequences. Removal of the antibiotic resistance markerfrom the transplastomic plants eliminates the metabolic burden imposedby the expression of the selectable marker gene and should also improvepublic acceptance of the transgenic crops. Additional applications ofthe CRE-lox site-specific recombination system are activation of plastidgene expression by deletion or inversion of plastid genome sequences andinduction of controlled cell death by deleting vital genes in the malereproductive tissue.

Although the use the CRE recombinase is exemplified herein, otherprokaryotic and eukaryotic site-specific recombinases would be equallysuitable for the elimination of the marker genes.

Recently, several prokaryotic and lower eukaryotic site-specificrecombination systems have been shown to operate successfully in highereukaryotes. In plant and animal cells functional site-specificrecombination systems from bacteriophages P1 (Cre-lox) Mu (Gin-gix), andfrom the inversion plasmids of Saccharomyces cerevisiae (FLP-frt)(Morris et al. 1991; O'Gorman et al. 1991; Lichtenstein and Barrena1993; Lyznik et al. 1993; Lyznik et al. 1995; Lyznik et al. 1996) andZygosaccharomyces rouxii (R—RS). In each of these systems, no additionalfactor aside from the recombinase and target sequences is required forrecombination. Reviewed in van Haaren and Ow, 1993. The CRE-loxPsite-specific recombination system of bacteriophage P1 has been studiedextensively in vitro and in E. coli (Craig 1988; Adams et al. 1992).Expression of the CRE protein (38.5 kDa) is sufficient to causerecombination between 34 bp loxP sites that consist of 13 bp invertedrepeats separated by 8 bp asymmetric spacer sequence. If there are twoloxP sites within a DNA segment, the result of the recombinationreaction depends on the relative position of the recombination sites. Ifthe recombination sites form a direct repeat, that if they are in thesame orientation, recombination results in deletion of the interveningDNA. If the recombination sites are in an inverted orientation,CRE-mediated recombination results in an inversion of the interveningDNA. The products of these reactions are shown in FIG. 1. The CREsite-specific recombination system has been employed for the eliminationof nuclear genes in a number of eukaryotic systems, including higherplants (Dale and Ow 1991; Russell et al. 1992; Srivastava et al. 1999).

Before the present invention, the efficiency of CRE-mediated eliminationof targeted plastid genes was unknown. To explore this system for thispurpose, CRE-mediated elimination of the codA gene encoding cytosinedeaminase (CD; EC 3.5.4.1) was assessed. Cytosine deaminase converts5-fluorocytosine (5FC) into 5-fluorouracil (5FU), the precursor of5-fluoro-dUMP. 5FC is lethal for CD-expressing cells due to irreversibleinhibition of thymidylate synthase by 5-fluoro-dUMP (Beck et al. 1972).Cytosine deaminase is absent in plants. Expression of the bacterial codain plastids renders cells sensitive to 5FC, while cells deficient intransgene expression are resistant (Serino and Maliga 1997). Thus, 5FCresistance could be used for positive identification of cells withCRE-induced coda deletion, even if such deletion events were relativelyrare. The test system of the present invention incorporates a coda genein the tobacco plastid genome between two directly oriented lox sites(>codA>). The transplastome was stable in the absence of CRE activity.However, highly efficient elimination of >codA> was triggered byintroduction of a nuclear-encoded plastid-targeted CRE.

EXAMPLE 1 Cre-Mediated Deletion of the Selectable Plastid Marker

Cre-mediated deletion of the selective plastid marker in the plastids oftobacco somatic cell is described in Example I. The selectable markerflanked by the lox sites is exemplified here by coda. However, it couldbe any other selectable and non-selectable marker gene, or any DNAsequence independent of information content flanked by lox sites in theplastid genome. Components of the test system are tobacco plantscarrying a coda coding region flanked by lox sites (>codA>). A secondcomponent of the test system is a nuclear gene encoding a plastidtargeted CRE-site specific recombinase. Deletion of a plastidencoded >codA> is achieved by introducing nuclear Cre into the nucleusof somatic (leaf) tobacco cells by Agrobacterium-mediatedtransformation. Alternatively, the nuclear encoded Cre gene may beintroduced by fertilization with pollen of an appropriateactivator-of-deletion strain. The nuclear Cre gene is subsequentlyremoved by segregation in the seed progeny.

Materials and Methods for the Practice of Example 1

The following materials and methods are provided to facilitate thepractice of Example 1.

Plastid codA with direct lox sites.

The codA gene is contained in a SacI-HindIII fragment. The gene map isshown in FIG. 2. PrrnloxD (Seq. ID No. 4) is a plastid rRNA operon(rrn16) promoter derivative. It is contained in a SacI-EcoRI fragmentobtained by PCR using oligonucleotides5′-GGGGAGCTCGCTCCCCCGCCGTCGTTCAATG-3′ (SEQ ID NO: 14) and5′-GGGAATTCATAACTTCGTATAGCATACATTATACGAAGTTAT GCTCCCAGAAATATAGCCA-3′(SEQ ID NO: 15) as primers and plasmid pZS176 (progenitor of plasmidpZS197; Svab and Maliga 1993) as a template. The promoter fragmentPrrnloxD contains a lox site at the 3′ end adjacent to the EcoRI site.The EcoRI-NcoI fragment contains the ribosome binding site from plasmidpZS176. The fragment was obtained by annealing the complementaryoligonucleotides 5′-AATTCGAAGCGCTTGGATAC AGTTGTAGGGAGGGATC-3′ (SEQ IDNO: 16) and 5′-CATGGATCCCTCCCTACAACTGTATCCAAGCGCTTCG-3′ (SEQ ID NO: 17).The codA coding region is contained in an NcoI-XbaI fragment (Serino andMaliga 1997). The TrbcLloxD (Seq. ID No. 5) is the rbcL 3′-untranslatedregion contained in an XbaI-HindIII fragment obtained by PCR usingoligonucleotides 5′-GGTCTAGATAACTTCGTATAATGTATGCTATACGAAGTTATAGACATTAGCAGATAAATT-3′ (SEQ ID NO: 18) and5′-GGGGGTACCAAGCTTGCTAGATTTTGTATTTCAAATCTTG-3′ (SEQ ID NO: 19) andplasmid pMSK48 (Khan and Maliga 1999) as template. TrbcLloxD contains alox site adjacent to the XbaI site in direct orientation relative to thelox site in the codA 5′UTR. The chimeric PrrnloxD:codA:TrbcLloxD genewas introduced into the tobacco plastid transformation vector pPRV111B(Zoubenko et al. 1994) as a SacI-HindIII fragment to obtain plasmidpSAC48.Plastid-targeted nuclear cre linked to a nuclear kanamycin resistancegene. Two plastid targeted nuclear cre genes were tested. The cre genein Agrobacterium binary vector pKO27 and pKO28 encode the CRErecombinase at its N terminus translationally fused with the pea Rubiscosmall subunit (SSU) chloroplast transit peptide (Timko et al. 1985) andtwenty two and five amino acids of the mature Rubisco small subunit,respectively. Both cre genes are contained in an EcoRI-HindIII fragment.The schematic map of the genes is shown in FIG. 3. The P2′ Agrobacteriumpromoter (Velten et al. 1984) (Sequence ID. No.9) is contained in anEcoRI-NcoI fragment. The P2′ promoter fragment was obtained by PCR usingoligonucleotides 5′-ccgaattcCATTTTCACGTGTGGAAGATATG-3′ SEQ ID NO: 20)and 5′-ccccatggtaggatcctatCGATTTGGTGTATCGAGATTGG-3′ (SEQ ID NO: 21) asprimers and plasmid pHC1 (Carrer et al. 1990) as template. PCRamplification introduced an EcoRI site at the 5′ end and ClaI, BamHI anda NcoI sites at the 3=end. A T introduced between the ClaI and the BamHIsites eliminates an ATG and introduces an in-frame stop codon (Sriraman2000). The Rubisco SSU transit peptides are included in BamHI-NcoIfragments. The pKO27 fragment (Pea SSU-TP22; Sequence ID No. 7) wasobtained by using oligonucleotides 5′-CCGGATCCAATTCAACCACAAGAACTAAC-3′(SEQ ID NO: 22) and 5′-GGGGCTAGCCATGGCAGGCCACACCTGCATGCAC-3′ (SEQ ID NO:23) as primers and plasmid pSSUpGEM4 as the template (Timko et al.1985). The pKO28 fragment (Pea SSU-TP5; Sequence ID No. 6) was obtainedby using oligonucleotides 5′-CCGGATCCAATTCAACCACAAGAACTAAC-3′ (SEQ IDNO: 22) and 5′-GGGGCTAGCCATGGTCAATGGGTTCAAATAGG-3′ (SEQ ID NO: 24) asprimers and plasmid pSSUpGEM4 as the template (Timko et al. 1985). Thecre coding region included in a NcoI-XbaI fragment (Sequence ID No. 3)was obtained by PCR amplification using the Cre15′-GGGGAGCTCCATGGCTAGCTCCAATTTACTGACCGTACAC-3′ (SEQ ID NO: 25) and Cre25′-GGGTCTAGACTAATCGCCATCCTCGAGCAGGCGCACCATTGC-3′ (SEQ ID NO: 26)oligonucleotides as primers and DNA isolated from Escherichia colistrain BNN132 (ATCC number 47059) as template. The presence of cre genein plant nuclear DNA was confirmed by PCR amplification with the Cre 1and Cre3 oligonucleotides. The sequence of Cre3 oligonucleotide is5′-TCAATCGATGAGTTGCTTC-3′ (SEQ ID NO:27). The Agrobacterium nosterminator (Tnos) is included in a XbaI-HindIII fragment (Svab et al.1990). The plastid targeted nuclear cre genes were introduced asEcoRI-HindIII fragments into the pPZP212 Agrobacterium binary vectors(Hajdukiewicz et al. 1994) to obtain plasmids pKO27 and pKO28 withtwenty two and five amino acids of the mature Rubisco SSU. A schematicmap of the Agrobacterium vectors is shown in FIG. 3.Transgenic plants. Plastid transformation using the biolistic protocol,selection of transplastomic tobacco clones (RMOP medium, 500 mg/Lspectinomycin dihydrochloride) and characterization of thetransplastomic clones by DNA gel blot analysis was described (Svab andMaliga 1993). Transformation with Agrobacterium vectors pKO28 or pKO27and regeneration of transformed tobacco plants has also been reported(Hajdukiewicz et al. 1994). Briefly, nuclear gene transformants wereselected by kanamycin resistance on RMOP shoot regeneration mediumcontaining 100 mg/L kanamycin and 500 mg/L carbenicillin. Kanamycinresistance of the shoots was confirmed by rooting on plant maintenance(RM) medium containing 100 mg/L kanamycin. Testing of 5FC cytotoxicitywas carried out on RMPO medium according to published procedures (Serinoand Maliga 1997).Transplastomic tobacco plants with a codA gene flanked by direct loxsites.Plastid transformation vector pSAC48 carries a codA gene in which twolox sites flank the coding region in a direct orientation. If the codAcoding region is deleted via the lox sites, a lox site flanked by thepromoter (Prrn) and terminator (TrbcL) are left behind. The selectivemarker in pSAC48, a pPRV111B vector derivative, is a spectinomycinresistance (aadA) gene (FIG. 2). Transformation with plasmid pSCAC48yielded a number of independently transformed transplastomic lines, ofwhich four were purified to the homoplastomic state: Nt-pSAC48-21A,Nt-pSAC48-16C, Nt-pSAC48-16CS and Nt-pSAC48-9A. These lines areconsidered identical other than they have been generated independently.A uniform population of transformed plastid genomes in thetransplastomic plants was verified by DNA gel blot analysis (see below).Nuclear-encoded plastid-targeted Cre genes.To activate deletion of the plastid >codA> gene we introduced anengineered cre gene into the nucleus of the transplastomic linesencoding a plastid-targeted CRE. Targeting of nuclear-encoded plastidproteins is by an N-terminal transit peptide (TP) cleaved off duringimport from the cytoplasm into plastids (Soll and Tien, 1998). To ensureplastid targeting of the CRE recombinase, it was translationally fusedwith the Rubisco small subunit (SSU) transit peptide (Timko et al.1985). Therefore, the product of the protein fusion is SSU-TP-CRE.Efficiency of import of chimeric proteins depends on the size of matureprotein N-terminus incorporated in the construct (Wasmann et al. 1986;Lubben et al. 1989). Two chimeric cre genes (Ssu-tp-cre) were prepared,one with 5 (vector pKO28) and one with 22 (plasmid pKO27) amino acids ofthe mature SSU N-terminus, encoding SSU-TP5-CRE and SSU-TP22-CRE,respectively. These genes are also referred to as Cre1 and Cre2,respectively (Table 1). The cre genes were expressed in the P2′ promoterand Tnos terminator cassettes in the Agrobacterium pPZP212 binary vectorwhich carries kanamycin resistance (neo) as a selectable marker (FIG.3).

Tobacco plant transformed with Ssu-tp5-cre (pKO37) and Ssu-tp22-cre(pKO36) were also obtained. In these plants the nuclear cre is expressedfrom the cauliflower mosaic virus 35S promoter (Seq. ID No. 10;Timmermans et al. 1990).

Nuclear Line Plastid genotype^(a) marker Wild-type trnV+ aadA− codA−Nt-pSAC48-21A trnV+ aadA+ codA+ Nt-pSAC48-16C Cre1-1 trnV+ aadA+ codA−neo trnV− aadA− codA− Cre1-2 trnV+ aadA+ codA− neo trnV− aadA− codA−Cre1-3 trnV+ aadA− codA− neo Cre1-4 trnV− aadA− codA− neo Cre1-10 trnV−aadA− codA− neo Cre2-1 trnV+ aadA+ codA− neo Cre2-2 trnV+ aadA+ codA−neo trnV+ aadA*+ codA− trnV− aadA− codA− Cre2-3 trnV+ aadA+ codA+ neotrnV+ aadA+ codA− trnV+ aadA*+ codA− trnV− aadA− codA− Cre2-4 trnV+aadA+ codA− neo Cre2-5 trnV+ aadA+ codA− neo Cre2-10 trnV+ aadA+ codA−neo trnV− aadA− codA− Cre1-100 trnV+ aadA− codA− neo Cre2-100 trnV+aadA− codA− neo Cre2-200 trnV+ aadA− codA− neo Cre2-300 trnV+ aadA−codA− neo ^(a)Presence or absence of plastid gene is indicated by + or−. Since the plastid trnV gene is deleted in some of the lines, thewild-type plastid genotype is trnV+ aadA− codA−.

Deletion of coda from the plastid genome in somatic cells.

To test the efficiency of CRE-mediated deletion in somatic cells, theSsu-tp-cre genes were introduced into the nucleus of thetransplastomic >codA> lines by cocultivation of Agrobacterium andtobacco leaf disks. Plants representing 11 individual Ssu-tp-creinsertion events have been characterized. Five lines (Cre1-derivatives)were obtained by transformation with Ssu-tp5-cre gene (vector pKO28) andsix lines (Cre2-derivatives) were obtained by transformation with theSsu-tp22-cre (vector pKO27) (Table 1). Deletion of coda was first testedin a DNA sample taken from one leaf of eleven kanamycin resistant shootsrepresenting an individual integration event of the nuclear Cre gene.Subsequently, 4 to 7 additional leaves were sampled from six shoots toconfirm that the result of the analysis is typical for the plant. Theinitial DNA samples were first screened for the loss of >codA> by PCRusing the O1/O2 primer pair complementary to sequences in the aadAcoding region N terminus and the coda promoter (FIG. 4A). Amplificationwith these primers yields a ˜0.7-kb fragment if >codA> is deleted and a˜2.0-kb fragment if the >codA> gene is still present. Ethidium bromidestained gels of PCR products in FIG. 5 indicate complete loss of >codA>in each of the samples. A perfect, reconstituted lox site between Prrnand TrbcL was confirmed in eight clones by PCR amplification of theregion with primers O1/O4 from the same DNA samples and directsequencing of the amplification product with primer O₂ (not shown). Intwo clones (Cre1-4, Cre1-10) a fragment is missing due to deletion ofaadA alongside with coda (see below). Plastid genome structure in theinitial DNA sample was determined by gel blot analysis of ApaI-EcoRVdigested total cellular DNA. The probes were the plastid targetingregion and the aadA and coda coding regions. The DNA gel blots are shownin FIG. 6. The maps of the parental genomes and deletion derivativesthat help to interpret these genomes are shown in FIG. 4. In the plastidtester strains expressing no CRE (Nt-pSAC48-21A, Nt-pSAC48-16C) allthree probes hybridized to the same 4.9-kb DNA fragment consistent withboth coda and aadA being present in all the plastid genome copies. Inthe SSU-TP-CRE expressing plants no 4.9-kb fragment was detectableindicating the dramatic speed by which the >codA> gene was eliminatedfrom the plastid genome. CRE-mediated deletion of >codA> via the loxsites yielded the 3.6-kb fragment detected in nine of the eleven clones.The 3.6-kb fragment was the only product detected in four clones, andwas present in a heteroplastomic population in five clones.Unanticipated was formation of a 1.4-kb ApaI-EcoRV fragment in fiveclones. DNA gel blot analysis confirmed that this fragment lacks bothcoda and aadA, and is smaller than the wild type ApaI-EcoRV fragment(1.9-kb). Direct sequencing of PCR products in this region confirmeddeletion of coda, aadA and trnV by homologous recombination via theduplicated Prrn promoter regions. One of the Prrn promoters is drivingcodA, the other is upstream of the rRNA operon at its native location.Deletion of trnV is the reason why the ApaI-EcoRV fragment derived fromthis region (1.4-kb) is smaller than the wild-type fragment (1.9-kb).The initial DNA samples were taken from one leaf of a plant obtained byrooting the shoot obtained after transformation with the Ssu-tp-cregenes. To confirm that the DNA samples extracted from the leaf weretypical for the plant, we have sampled several more leaves from the sameplants (FIG. 7). In four clones coda was excised by CRE via the loxsites, and the shoots were homoplastomic for the deleted genome. Two ofthese, Cre1-3 and Cre2-4 were further characterized by testing seven andfour additional leaves of the same plants, respectively. DNA gel blotanalysis of these samples confirmed a uniform deletion of >codA> fromall genome copies. These plants are the desired final products carryingthe desired plastid transgenes and lacking the undesirable selectivemarker. These plants and their progeny can be used directly for theproduction of recombinant proteins as they are free from the selectablemarker gene. Furthermore, these plants are a source of engineeredchloroplasts for introduction into breeding lines by sexual crossing.The seed progeny of the plants is segregating for the Ssu-tp-creactivator gene. Plants with the desired chloroplasts but lacing theactivator gene can be identified by PCR testing for cre sequences.Alternatively, individuals lacking cre can be identified in the seedprogeny by sensitivity to kanamycin, since the Ssu-tp-cre genes in thepKO27 and pKO28 Agrobacterium vectors are physically linked to kanamycinresistance (neo gene; FIG. 3). In two clones, Cre1-4 and Cre1-10,deletion of trnV (encoding tRNA-Val^(GAC)), aadA and codA occurred byhomologous recombination via the duplicated Prrn promoter region. TheCre1-10 plant is homoplastomic for the deletion based on probing sevenadditional leaves (FIG. 7). Apparently, the one remaining trnV geneencoding tRNA-Val^(UAC) is sufficient for the translation of all valinecodons, or there is import of tRNA-Val^(GAC) from the cytoplasm. In theCre1-4 clone some of the leaves (two out of four) contained residualgenome copies with trnV and aadA.

In five clones the initial DNA samples contained more than one type ofplastid genome copies. Mixed populations of plastid genome populationswere confirmed in all parts of the plants by testing additional leaves(FIG. 7). Genetically stable coda deletion lines can be obtained fromthese heteroplastomic plants by testing plants regenerated from singlesomatic cells or individual seedlings in a segregating seed progeny.

Deletion of coda from the plastid genome in the seed progeny.

CRE-mediate deletion of the negative plastid marker coda in somaticcells was described in the previous section. Deletion of the plastidmarker gene in the somatic cells of the transplastomic plants, withoutgoing though a sexual cycle, is highly desirable to accelerate theproduction of marker-free transplastomic plants. However, this approachis feasible only if there is a system for tissue culture and plantregeneration from somatic cells. Such system is unavailable for theeconomically important cereal crops rice and maize. As an alternative totransformation of somatic cells, we developed CRE activator linescarrying a nuclear-encoded plastid-targeted Cre to be used as the sourceof Cre gene when used as a pollen parent. The tobacco CRE activatorlines were obtained by transforming the nucleus of wild-type plants withSSU-TP-CRE constructs. Lines in which the Cre is linked to a nuclearkanamycin resistance gene in a wild-type cytoplasm are Cre1-100,Cre-2-100, Cre2-200 and Cre2-300 (Table 1). To activate deletionof >codA> in the seed progeny, tester plants Nt-pSAC48-21A andNt-pSAC48-16C were emasculated to prevent self fertilization, andfertilized with pollen from the Cre2-200 and Cre2-300 activator lines.The activator lines are primary transgenic plants (T₀) segregating forthe Ssu-tp-cre gene. Therefore, a proportion of the seed progeny derivedfrom the cross will have the activator genes while others will not. Ifthe coda gene is present, the O1/O2 primer pair marked in FIG. 4amplifies a 2.0-kb fragment. If the coda gene is absent, the sameprimers will amplify a 0.7-kb fragment. PCR analysis shown in FIG. 8confirmed CRE-mediated deletion of >codA> in seedlings. The Cre1-100,Cre2-100 and Cre2-300 activator lines are apparently expressing CREefficiently, indicated by the presence of only of the 0.7-kb fragment inseedlings carrying the nuclear cre gene. In seedlings with no cresequence the same primers amplified the 2.0-kb coda-containing fragment.Interestingly, cre+ seedlings from the cross with Cre2-200 contained amixed population of coda containing (2.0-kb) and coda-deleted (0.7-kb)fragments indicating less efficient CRE-induced deletion of >codA>.Thus, expression level and tissue specificity of the two nuclearSsu-tp22-cre genes are characteristic for the individual transformationevents. CRE activity of Cre1-100, Cre2-100 and Cre2-300 activator linesis more suitable for rapid elimination of >codA> in a cross than theCre2-200 line. It is undesirable to maintain the Ssu-tp-cre activatorgenes in the production lines. However, these are encoded in thenucleus, and can be separated from the transgenic chloroplasts in thenext seed progeny. Linkage of Ssu-tp-cre to the nuclear kanamycinresistance gene facilitates identification of seedlings lacking cre in asegregating seed population.CRE site-specific recombinase for deletion of plastid DNA sequences.Biolistic transformation of tobacco leaves always yields shootscontaining a mixed population of plastid genome copies. A mixedpopulation of plastid genome copies is determined by DNA gel blotanalysis (Carrer et al. 1993; Svab and Maliga 1993; Carrer and Maliga1995) and can be visualized in UV light when expressing the greenfluorescence protein in plastids (Khan and Maliga 1999). Homoplastomic,genetically stable plants are obtained during a second cycle of plantregeneration from the leaves of the regenerated plants or in the seedprogeny. The cells of the >codA> tester strains carry a uniformpopulation of plastid genome copies. Thus, the Ssu-tp-cre is introducedinto the nuclear genome of a cell that is homoplastomic for >codA>. Itwas expected that the regenerated shoots would contain a mixedpopulation of plastid genome copies. Instead, all plastid genome copieslack >codA>, an evidence for the enormous selection pressure by CREactivity against plastid genome copies that carry two lox sites. It isimportant that deletion of >codA> occurs in the absence of selectionagainst >codA> by exposure to 5-fluorocytosine. Virtually completeelimination of >codA> may also be obtained when CRE activity isintroduced by crossing, using pollen of an appropriate deletionactivator strain. Deletion of the selectable marker in somatic cells isthe preferred choice over elimination of the marker in the seed progeny.The most important advantage is time saving. Introduction of Ssu-tp-creinto the nucleus of somatic cells requires only three to six weeks;Ssu-tp-cre segregates out in the first seed progeny. In contrast,introduction and elimination of Ssu-tp-cre takes one additional seedprogeny, about three months.

Interestingly, genome copies with one lox site or no lox site(wild-type) are stable in CRE-expressing cells. Instability of genomeswith two lox sites may be due to formation of linear ends during theexcision process. The linear ends may then re-circularize by homologousrecombination via the Prrn promoter sequences yielding thetrnV-aadA-codA deletion derivatives.

CRE engineering. Although CRE is a prokaryotic protein, it naturallycarries a nuclear localization signal (NLS) that targeted a CRE-GFPfusion protein to the nucleus in mammalian cells. The NLS sequencesoverlap the DNA binding regions and the integrity of this region isimportant for DNA recombinase activity (Le et al. 1999). We targeted thenewly-synthesized TP-CRE protein to plastids using a plastid-targetingtransit peptide (TP). The TP is localized at the N terminus of plastidproteins and is cleaved off during import from the cytoplasm intoplastids (Soll and Tien, 1998). Therefore, we translationally fused aplastid transit peptide with CRE to direct its import from the cytoplasmto plastids. Translational fusion yielded a protein with an N-terminalplastid targeting signal and an internal nuclear localization signal.Efficient CRE-mediated deletion of plastid-encoded coda genes indicatestargeting of SSU-TP-CRE to plastids. When two potential targetingsequences are present, in general one of them out-competes the other(Small et al. 1998). N-terminal organelle targeting sequences normallydominate the second internal localization signal. For example, the70-kDa heat shock protein of watermelon cotyledons that carry N-terminalplastidal and internal glyoxysomal targeting sequences are exclusivelytargeted to plastids. Proteins are localized to glyoxysomes only in theabsence of the plastidal presequence (Wimmer et al. 1997). The tRNAmodification enzymes contain information for both mitochondrial(N-terminal extension) and nuclear targeting. The enzyme with theN-terminal extension is targeted to mitochondria and only the short formlacking the N-terminal extension is targeted to the nucleus (Small etal. 1998). It was fortunate, that the Rubisco SSU N-terminal transitpeptide dominated the CRE nuclear localization signals and the TP-CREfusion protein was directed to plastids (chloroplasts). A secondproperty that is important for the present invention is maintenance ofrecombinase activity when CRE is fused with proteins or peptides at itsN and C termini. N-terminal fusion of CRE with the E. coli maltosebinding protein did not interfere with recombinase function (Kolb andSiddell 1996). CRE was also shown to accept a C-terminal fusion with GFP(Le et al. 1999) as well as an 11-amino-acid epitope to the herpessimplex virus (HSV) glycoprotein D coat protein. The epitope tagfacilitates detection of CRE expression in vitro and in vivo usingimmunofluorescent labeling with a commercially available antibody(Stricklett et al. 1998). Apparently, the five and 22 amino acids thatare left behind after processing of the SSU-TP5-CRE and SU-TP22-CREproteins did not interfere with CRE function.Dominant negative selection markers for positive identification ofdeletion derivatives. A practical application of the present inventionis the removal of selectable marker genes from the transformed plastidgenome. In tobacco, the excision process mediated by the CRE constructsdescribed herein is so efficient that the >codA> deletion derivativescan be identified in the absence of 5FC selection. However, in othercrops CRE-mediated excision of marker genes may be less efficient. Inthese species, the positive selective marker (aadA) may be fused with adominant negative selective marker using linker peptides as described inthe literature (Khan and Maliga 1999) or the positive and negativemarker genes may be combined in a dicistronic operon (Staub and Maliga1995). Dominant negative selection markers allow normally non-toxiccompounds to be used as toxic agents, so that cells which express thesemarkers are non-viable in the presence of the compound, while cells thatdon't carry them are unaffected. For example, cytosine deaminase isabsent in plants. Expression of coda, encoding cytosine deaminase (CD;EC 3.5.4.1), in plastids renders tissue culture cells and seedlingssensitive to 5FC, facilitating direct identification of clones lackingthis negative selective marker (Serino and Maliga 1997). Cytosinedeaminase converts 5-fluorocytosine (5FC) into 5-fluorouracil (5FU), theprecursor of 5-fluoro-dUMP. 5FC is lethal for CD-expressing cells due toirreversible inhibition of thymidylate synthase by 5-fluoro-dUMP (Becket al. 1972). We have found that seedlings and plant tissuesexpressing >codA> were sensitive to 5FC. Seedlings lacking coda could bereadily identified by 5FC resistance. Thus, the constructs describedhere are suitable to express cytosine deaminase at sufficiently highlevels to be useful to implement a negative selection scheme.

Alternative negative selective markers can be obtained by adaptation ofsubstrate-dependent negative selection schemes described for nucleargenes. Such negative selection schemes are based on resistance toindole, napthyl, or naphtalene acetamide (Depicker et al. 1988;Karlin-Neumann et al. 1991; Sundaresan et al. 1995), chlorate (Nussaumeet al. 1991), kanamycin (Xiang and Guerra 1993) and 5-fluorocytosine(5FC) (Perera et al. 1993; Stougaard 1993).

EXAMPLE 2 Cr-Mediated Inversion of Plastid DNA Sequences

If the lox sites in bacteria are in an inverted orientation,CRE-mediated recombination results in an inversion of the interveningDNA. We have tested, whether the CRE-mediated inversion reaction alsooccurs in plastids of higher plants containing DNA sequences flanked byinverted lox sites. This was assessed using a kanamycin-resistance(>neo<) coding region in an inverted orientation relative to thepromoter (FIG. 9). In this construct the non-coding strand of neo istranscribed and the plants are kanamycin sensitive. The >neo< codingregion is flanked by inverted lox sites. CRE-mediated inversion of thesequences reverses neo orientation resulting in the transcription of thesense strand and expression of kanamycin resistance. Inversion of theplastid-encoded >neo< coding region may be achieved by multipleapproaches. One approach is to introduce a nuclear Cre into the nucleusof somatic tobacco cells, e.g., leaf, by Agrobacterium-mediatedtransformation. A second approach is introduction of the nuclear-encodedCre gene by fertilization with pollen of an appropriateactivator-of-inversion strain. Additional approaches are to provideCRE-activity via the incorporation of chemically inducible promoter intothe construct, or to transiently express CRE from a nuclear ofchloroplast construct.

Materials and Methods for the Practice of Example 2

Plastid neo gene with inverted lox sites. The neo gene is contained in aSacI-HindIII fragment. The gene map is shown in FIG. 8. PrrnloxI (Seq.ID No. 1) is a plastid rRNA operon (rrn16) promoter derivative. It iscontained in a SacI-XbaI fragment obtained by PCR using oligonucleotides5′-ggggagctcGCTCCCCCGCCGTCGTTCAATG-3′ (SEQ ID NO: 14) and5′-ggtctagataacttcgtatagcatacattatacgaagttatGCTCCC AGAAATATAGCCA-3′ (SEQID NO: 28) as primers and plasmid pZS176 (progenitor of plasmid pZS197;Svab and Maliga 1993) as a template. The promoter fragment PrrnloxIcontains a lox site at the 3′ end adjacent to the XbaI site. The neocoding region is contained in an NcoI-XbaI fragment derived from plasmidpHC62. The neo sequence in plasmid pHC62 is identical with the neosequence shown in FIG. 28B, U.S. Pat. No. 5,877,402. The EcoRI-NcoIfragment contains the ribosome binding site from plasmid pZS176. Thefragment was obtained by annealing the complementary oligonucleotides5′-AATTCGAAGCGCTTGGATACAGTTGTAGGGAGGGATC-3′ (SEQ ID NO: 16) and5′-CATGGATCCCTCCCTACAACTGTATCCAAGCGCTTCG-3′(SEQ ID NO: 17). TheTrbcLloxI (Seq. ID No. 2) is the rbcL 3′-untranslated region containedin an EcoRI-HindIII fragment obtained by PCR using oligonucleotides5′-gggaattcataacttcgtatagcatacattatac gaagttatAGACATTAGCAGATAAATT-3′(SEQID NO: 29) and 5′-gggggtaccaagcttgCTAGATTTTGTATTTCAAATCTTG-3′ (SEQ IDNO: 19) and plasmid pMSK48 (Khan and Maliga 1999) as template. TrbcLloxIcontains a lox site adjacent to the EcoRI site in an invertedorientation relative to the lox site in PrrnloxI. The chimericPrrnloxI:neo:TrbcLloxI gene was introduced into the tobacco plastidtransformation vector pPRV111B (Zoubenko et al. 1994)

Plastid-targeted nuclear cre linked to a nuclear gentamycin resistance(aacC1) gene. The plastid targeted nuclear cre genes were introduced asEcoRI-HindIII fragments into the pPZP222 Agrobacterium binary vectorswhich carry a plant-selectable gentamycin resistance gene (Hajdukiewiczet al. 1994) to obtain plasmids pKO30 and pKO31 with twenty two and fiveamino acids of the mature Rubisco SSU. The map of the Agrobacteriumvectors is identical with the one shown in FIG. 3. other than they carrya gentamycin resistance gene.Transplastomic tobacco plants with a neo gene flanked by inverted loxsites.Plastid transformation vector pSAC38 with the inverted >neo< gene isshown in FIG. 9. The inverted >neo< gene was introduced into plastids byselection for spectinomcyin resistance (aadA) encoded in the vector. Twoindependently transformed lines were purified to the homoplastomicstate: Nt-pSAC38-9A and Nt-pSAC38-10C. The homoplastomic state wasconfirmed by DNA gel blot analysis.Nuclear-encoded plastid-targeted Cre genes.

Plant activator lines in which Ssu-tp-cre is linked to a nuclearkanamycin resistance gene have been described in Example 1. The plastidmarker to test CRE-activated inversion described in Example 2 utilizes akanamycin resistance gene. Kanamycin resistance conferred by the plastidgene due to CRE-mediated inversion could not be distinguished fromkanamycin resistance conferred by the marker gene of the Agrobacteriumbinary vector that was used to introduce the nuclear cre. Therefore, wehave constructed activator strains in which Ssu-tp-cre is linked togentamycin resistance. The Ssu-tp22-cre gene linked to the nucleargentamycin resistance is the Cre3 strain and the Ssu-tp5-cre gene linkedto gentamycin resistance is the Cre4 strain.

Inversion of >neo< in the plastid genome of somatic cells.

The nuclear cre genes were introduced into the chloroplast >neo< testerstrains by cocultivation of tobacco leaves with the Agrobacteriumstrains and selection for gentamycin resistance (100 mg/L). Digestion oftotal cellular DNA with BamHI and probing with the plastid targetingregion (ApaI-EcoRV fragment, FIG. 4) hybridizes to 1.8-kb and a 3.8-kbfragments in the parental Nt-pSAC38-10C lines (FIG. 10). Activation byCRE in lines Cre3-3 and Cre4-5 created a mixed population of >neo< genesrepresenting the original and inverted orientations detected as theoriginal 3.8-kb and 1.8-kb and the newly created 4.6-kb and 0.9-kbhybridizing fragments. Lines carrying the cre and an approximatelywild-type size fragment are aadA-neo deletion derivatives, similar tothose shown in FIG. 4. Thus, it appears that CRE mediated inversion vialox sites creates increased local recombination frequencies that leadsto deletion of the transgenes via the short direct repeats of Prrnpromoters.

Controlling inversion via lox sites by CRE activity.

Here we describe constructs for CRE-mediated inversion of plastid genomesegments flanked by inverted lox sites. Inversion of the sequences isindependent of the encoded genetic information and relies only on CREactivity. CRE activity may be provided transiently, by expression inplastids from plastid signals described in U.S. Pat. No. 5,877,402, orfrom nuclear genes encoding a plastid-targeted CRE. Suchplastid-targeted CRE constructs are described in Example 1, for examplethe Ssu-tp5-cre or Sssu-tp22-cre genes. Alternative approaches toprovide CRE activity are stable incorporation of a plastid-targetednuclear Cre into the nucleus of somatic (leaf) cells byAgrobacterium-mediated, PEG induced or biolistic transformation or byfertilization with pollen from a transformed plant. The Agrobacterium P2promoter and cauliflower mosaic virus 35S promoter exemplified here areconstitutive promoters. Regulated expression of CRE may be important forcertain applications. Developmentally timed expression may be obtainedfrom promoters with tissue specific activity. Regulated expression ofCRE may be obtained from chemically induced nuclear gene promotersresponding to elicitors, steroids, copper or tetracycline (reviewed in;Gatz et al. 1992; Mett et al. 1993; Aoyama and Chau 1997; Gatz 1997;Martinez et al. 1999; Love et al. 2000) and described in U.S. Pat. No.5,614,395.

Controlled Expression of Deleterious Gene Products

There are a variety of valuable heterologous proteins that interferewith plastid metabolism. For example, certain proteins may be insertedinto photosynthetic membranes and interfere with photosynthesis. Thisproblem can be circumvented by first growing the plants to maturity,then activating production of the deleterious protein by chemicallyinducing CRE expression. CRE, in turn, will make the gene expressible bylox-mediated inversion of the coding region.

The molecular tools necessary for the construction of such plastid genesare described in present application. In case of the monocistronicinversion vector the gene of interest (goi) is flanked by inverted loxsites and is introduced by linkage with aadA (FIG. 12). The selectablemarker (aadA) coding region is the first reading frame, and is expressedfrom the promoter. The goi reading frame is the second coding region,and it is not expressed as it is in an inverted orientation relative tothe promoter. Expression of goi is induced by CRE-mediated inversion ofthe goi coding region, as described for >neo< in Example 2 and is shownin FIG. 12.

The dicistronic lox inversion vector is shown in FIG. 13. In this casethe inverted lox sites flank both aadA and goi. The selectable marker(aadA) coding region is expressed from the promoter. The goi readingframe is not expressed as it is in an inverted orientation relative tothe promoter. Expression of goi is induced by CRE-mediated inversion ofthe aadA-goi containing region that results in simultaneous expressionof goi and inactivation of aadA.

The presence of two lox sites may destabilize the plastid genome thatleads to CRE-independent deletion of plastid genome sequences. However,it appears that CRE activity by itself is not mutagenic, and the plastidgenomes are stable if only one lox site is present. Mutant lox sitesthat are efficiently excised but recombine into excision resistant siteshave been described (Hoess et al. 1982; Albert et al. 1995). Such loxsites would mediate efficient inversion, but the new lox sites would beresistant to additional cycles of CRE activation. Providing only a shortburst of CRE activation using a chemically induced promoter couldfurther refine the expression system.

EXAMPLE 3 CRE-Mediated Deletion to Obtain Marker Free TransplastomicPlants and for High Level Expression of the Recombinant Proteins

Plastid loxP vectors in this section are described for CRE-mediatedexcision of selective markers in transplastomic plants. Since excisionof sequences between directly oriented lox sites is very efficient,variants of the same vectors can be used for CRE-activated expression ofrecombinant proteins. A family of plastid vectors with suitablypositioned lox sites is shown schematically in FIG. 14 through FIG. 17.

The map of the basic tobacco plastid lox deletion vector is shown inFIG. 14. It contains (a) two directly oriented lox sites separated by aunique BglII cloning site and (b) an adjacent polycloning site. Thesesequences (Seq. ID No. 11) are inserted into the ScaI site plastidrepeat vector pPRV100 (U.S. Pat. No. 5,877,402; Zoubenko et al. 1994).Suitable marker genes (aadA, neo or kan, bar, glyphosate resistance,bromoxynil resistance) for insertion into the BglII site have beendescribed in U.S. Pat. No. 5,877,402, WO 00/07421 and WO 00/03022.

The map of the tobacco plastid lox >aadA> deletion vector is shown inFIG. 15. It is the basic lox deletion vector with an aadA gene clonedinto the BglII sites oriented towards the rrn operon.

Maps of constitutive lox dicistronic deletion vectors are shown in FIG.16 through FIG. 18. This dicistronic design enables simultaneousexpression of both the first and the second open reading frames. Theselectable marker designed for excision may be encoded in the first(FIG. 16) or second (FIG. 17, FIG. 18) open reading frames. Since aminimally 34 bp lox site is located between the two reading frames, boththe marker gene (aadA) and the gene of interest have their own leadersequence to facilitate translation (FIG. 16, FIG. 17). Translationalcoupling may also be feasible if the lox site is incorporated in themarker gene coding region N terminus (FIG. 18). DNA sequence ofpromoter-lox constructs shown in FIG. 16 is set forth in Seq. ID No. 1.Promoters and promoter-leader combinations suitable to promotehigh-level protein expression in plastids are described in EuropeanPatent Applications WO 00/07421, WO 97/06250 and WO 98/55595. Sequencessuitable for directly oriented lox sites are given in Seq. ID No. 11.Translational coupling between a gene of interest and the downstreamaadA is shown in FIG. 18. There are multiple ways of achievingtranslational coupling between adjacent genes (Baneyx 1999). Oneapproach is incorporation of a properly spaced ribosome binding-site inthe upstream gene's coding region (Schoner et al. 1986; Omer et al.1995). An example for a suitable sequence directly upstream of thetranslation initiation codon (ATG) would be G-GAG-GAA-TAA-CTT-ATG. Aspecific example for the use of the sequence is translational couplingbetween a bar (suitable source described in European Patent ApplicationWO 00/07421) and a downstream aadA are given in Seq. ID No. 12. NoteSalI site downstream of AUG incorporated to facilitate engineering theBglII-SalI region and the directly oriented lox sites in the aadA codingregion and downstream of aadA. The sequence is given for a BglII-SpeIfragment. The BglII site is within the bar coding region; the SpeI siteis downstream of the second lox site, as marked in FIG. 18. If aC-terminal extension to create a ribosome binding site is unacceptable,a suitable sequence may be obtained by silent mutagenesis of the codingregion at the third codon position. Variants of plastid ribosome bindingsites have been catalogued (Bonham-Smith and Bourque 1989)

A tobacco inducible lox deletion vector is shown in FIG. 19. The markergene (aadA) is encoded in the first reading frame, followed by a silentgoi lacking the translation initiation codon (ATG) and the 5′untranslated leader. Expression of the goi frame is triggered by aadAexcision that results in translational fusion of the aadA N-terminalregion with the goi. After aadA excision the goi mRNA is translated fromthe aadA translation control signals, the 5′ UTR and AUG. DNA sequenceof the SacI-NheI fragment is given in Seq. ID. No. 13. The Prrnpromoter-atpB translational control region is described in EuropeanPatent Application WO 00/07421. The aadA construct has twodirectly-oriented lox sites: one in the coding region N-terminus and onedownstream of aadA to facilitate CRE-mediated excision of the markergene.

EXAMPLE 4 Deletion of Vital Plastid Genes to Obtain Cytoplasmic MaleSterility

U.S. Pat. No. 5,530,191 provides a cytoplasmic male sterility (CMS)system for plants, which is based on modification of the plastid genome.The CMS system comprises three transgenes: a “plastid male sterility”gene that causes plastid and cellular disablement of the anther tissue,and two nuclear genes that regulate the expression of the plastid gene.An important feature of the system is developmentally timed cellulardeath based on the expression, or the lack of the expression, of aplastid gene. As one specific approach to induce developmentally timedablation of anther tissue we describe CRE-mediate excision of essentialplastid genes via directly oriented lox sites.

The number of genes encoded by the plastid genome is about 120. Some ofthe genes are non-essential and may be inactivated by targeted genedisruption without a major phenotypic consequence. Good examples are theplastid ndh genes (Burrows et al. 1998; Shikanai et al. 1998) or thetrnV gene the deletion of which has been described in Example 1.Excision of these genes is unlikely to cause cell ablation. Thephotosynthetic genes are essential for survival under field conditions.However, pigment deficient, non-photosynthetic plants can be maintainedas long as they are grown on a sucrose-containing medium, or are graftedonto photosynthetically active wild-type (green) plants (Kanevski andMaliga 1994). Some of the house-keeping genes, such as the genesencoding the plastid multisubunit RNA polymerase are essential forphotosynthetic growth, but not for survival (Allison et al. 1996). Thus,deletion of these genes is not suitable to trigger cell death. Only arelatively small number of plastid genes have proven to be essential forviability. The essential nature of the genes was recognized by the lackof homoplastomic cells in gene disruption experiments indicating thatthe loss of these genes results in cellular death. Cellular death due tolack of plastid function is understandable, as plastids are the site ofthe biosynthesis of amino acids, several lipids and are required fornitrate assimilation. Examples of plastid genes essential for cellularsurvival are the clpP protease subunit gene (Huang et al. 1994), ycf1and ycf2, the two largest plastid-encoded open reading frames (Drescheret al. 2000).

To induce cellular death by CRE-mediated excision, directly oriented loxsites can be incorporated in the plastid genome flanking essentialgenes, as shown for clpP in FIG. 20. The clpP gene has two large introns(807 bp and 637 bp) and the region can be conveniently cloned as aSalI-SphI fragment. The selectable marker aadA is inserted into a KpnIrestriction site created by PCR mutagenesis downstream of clpP Exon 3,oriented towards rps12 Exon I. One of the lox sites is engineered nextto the aadA gene, the second lox site is inserted in Intron I. Cellulardeath is induced by activation of the nuclear Cre gene as described inU.S. Pat. No. 5,530,191. It is necessary to use a selective marker, suchas aadA to introduce the lox sites into the plastid genome. The aadAgene can subsequently eliminated using a second, independent sitespecific recombinase such as FRT via the frt sites engineered into thetransformation vector shown in FIG. 20.

Alternative targets for CRE-mediated deletion in a CMS system are theessential ribosomal protein genes such as rp123, the ribosomal RNAoperon (for insertion sites see; Staub and Maliga 1992; Zoubenko et al.1994) and the ycf1 and ycf2 genes (Drescher et al. 2000)

The following sequences are referred to throughout the specification andfacilitate the practice of the present invention.

SEQ. No. 1: PrrnloxI. sequence

gagctcGCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTGGGATTGACGTGAGGGGGCAGGGATGGCTATATTTCTGGGAGCataacttcgtataatgtatgctatacgaagttatctaga

SEQ. No. 2: TrbcLloxI. sequence

gaattcataacttcgtatagcatacattatacgaagttatAGACATTAGCAGATAAATTAGCAGGAAATAAAGAAGGATAAGGAGAAAGAACTCAAGTAATTATCCTTCGTTCTCTTAATTGAATTGCAATTAAACTCGGCCCAATCTTTTACTAAAAGGATTGAGCCGAATACAACAAAGATTCTATTGCATATATTTTGACTAAGTATATACTTACCTAGATATACAAGATTTGAAATACAAAATCTAGcaagcttggtaccSEQ. No. 3: cre coding region. sequence

gagctccATGgctagcTCC AATTTACTGA CCGTACACCA AAATTTGCCT GCATTACCGGTCGATGCAAC GAGTGATGAG GTTCGCAAGA ACCTGATGGA CATGTTCAGG GATCGCCAGGCGTTTTCTGA GCATACCTGG AAAATGCTTC TGTCCGTTTG CCGGTCGTGG GCGGCATGGTGCAAGTTGAA TAACCGGAAA TGGTTTCCCG CAGAACCTGA AGATGTTCGC GATTATCTTCTATATCTTCA GGCGCGCGGT CTGGCAGTAA AAACTATCCA GCAACATTTG GGCCAGCTAAACATGCTTCA TCGTCGGTCC GGGCTGCCAC GACCAAGTGA CAGCAATGCT GTTTCACTGGTTATGCGGCG GATCCGAAAA GAAAACGTTG ATGCCGGTGA ACGTGCAAAA CAGGCTCTAGCGTTCGAACG CACTGATTTC GACCAGGTTC GTTCACTCAT GGAAAATAGC GATCGCTGCCAGGATATACG TAATCTGGCA TTTCTGGGGA TTGCTTATAA CACCCTGTTA CGTATAGCCGAAATTCCCAG GATCAGGGTT AAAGATATCT CACGTACTGA CGGTGGGAGA ATGTTAATCCATATTGGCAG AACGAAAACG CTGGTTAGCA CCGCAGGTGT AGAGAAGGCA CTTAGCCTGGGGGTAACTAA ACTGGTCGAG CGATGGATTT CCGTCTCTGG TGTAGCTGAT GATCCGAATAACTACCTGTT TTGCCGGGTC AGAAAAAATG GTGTTGCCGC GCCATCTGCC ACCAGCCAGCTATCAACTCG CGCCCTGGAA GGGATTTTTG AAGCAACTCA TCGATTGATT TACGGCGCTAAGGATGACTC TGGTCAGAGA TACCTGGCCT GGTCTGGACA CAGTGCCCGT GTCGGAGCCGCGCGAGATAT GGCCCGCGCT GGAGTTTCAA TACCGGAGAT CATGCAAGCT GGTGGCTGGACCAATGTAAA TATTGTCATG AACTATATCC GTAACCTGGA TAGTGAAACA GGGGCAATGGTGCGCCTGCT cGAgGATGGC GATTAGtctagaSEQ. No. 4: PrrnloxD. SequencegagctcGCTCCCCCGCCGTCGTTCAATGAGAATGGATAAGAGGCTCGTGGGATTGACGTGAGGGGGCAGGGATGGCTATATTTCTGGGAGCataacttcgtataatgtatgctatacgaagttatgaattcSEQ. No. 5: TrbcLloxD. sequencetctagataacttcgtataatgtatgctatacgaagttatAGACATTAGCAGATAAATTAGCAGGAAATAAAGAAGGATAAGGAGAAAGAACTCAAGTAATTATCCTTCGTTCTCTTAATTGAATTGCAATTAAACTCGGCCCAATCTTTTACTAAAAGGATTGAGCCGAATACAACAAAGATTCTATTGCATATATTTTGACTAAGTATATACTTACCTAGATATACAAGATTTGAAATACAAAATCTAGcaagcttggtacc

SEQ. No. 6: Pea ssuTP5. sequence ccggatccAA TTCAACCACA AGAACTAACAAAGTCAGAAA AATGGCTTCT ATGATATCCT CTTCCGCTGT GACAACAGTC AGCCGTGCTTCTAGGGTGCA ATCCGCGGCA GTGGCTCCAT TCGGCGGCCT GAAATCCATG ACTGGATTCCCAGTGAAGAA GGTCAACACT GACATTACTT CCATTACAAG CAATGGTGGA AGAGTAAAGTGCATGCAGGT GTGGCCTgcc atggctagc SEQ. No. 7: Pea ssuTP22. sequenceccggatcc AA TTCAACCACA AGAACTAACA AAGTCAGAAA AATGGCTTCT ATGATATCCTCTTCCGCTGT GACAACAGTC AGCCGTGCTT CTAGGGTGCA ATCCGCGGCA GTGGCTCCATTCGGCGGCCT GAAATCCATG ACTGGATTCC CAGTGAAGAA GGTCAACACT GACATTACTTCCATTACAAG CAATGGTGGA AGAGTAAAGT GCATGCAGGT GTGGCCTCCA ATTGGAAAGAAGAAGTTTGA GACTCTTTCC TATTTGCCAC CATTGACCat ggctagc SEQ. No. 8: PeassuTP23. sequence ccggatccAA TTCAACCACA AGAACTAACA AAGTCAGAAA AATGGCTTCTATGATATCCT CTTCCGCTGT GACAACAGTC AGCCGTGCTT CTAGGGTGCA ATCCGCGGCAGTGGCTCCAT TCGGCGGCCT GAAATCCATG ACTGGATTCC CAGTGAAGAA GGTCAACACTGACATTACTT CCATTACAAG CAATGGTGGA AGAGTAAAGT GCATGCAGGT GTGGCCTCCAATTGGAAAGA AGAAGTTTGA GACTCTTTCC TATTTGCCAC CATTGACCAG AGATCAGTTGgctagcgg SEQ. No. 9: P2 promoter sequence gaattCATTT TCACGTGTGGAAGATATGAA TTTTTTTGAG AAACTAGATA AGATTAATGA ATATCGGTGT TTTGGTTTTTTCTTGTGGCC GTCTTTGTTT ATATTGAGAT TTTTCAAATC AGTGCGCAAG ACGTGACGTAAGTATCTGAG CTAGTTTTTA TTTTTCTACT AATTTGGTCG TTTATTTCGG CGTGTAGGACATGGCAACCG GGCCTGAATT TCGCGGGTAT TCTGTTTCTA TTCCAACTTT TTCTTGATCCGCAGCCATTA ACGACTTTTG AATAGATACG CTGACACGCC AAGCCTCGCT AGTCAAAAGTGTACCAAACA ACGCTTTACA GCAAGAACGG AATGCGCGTG ACGCTCGCGG TGACGCCATTTCGCCTTTTC AGAAATGGAT AAATAGCCTT GCTTCCTATT ATATCTTCCC AAATTACCAATACATTACAC TAGCATCTGA ATTTCATAAC CAATCTCGAT ACACCAAATC GATaggatcctaccatgg SEQ. No. 10: 35S promoter sequence AAGCTTGCCA ACATGGTGGAGCACGACACT CTCGTCTACT CCAAGAATAT CAAAGATACA GTCTCAGAAG ACCAAAGGGCTATTGAGACT TTTCAACAAA GGGTAATATC GGGAAACCTC CTCGGATTCC ATTGCCCAGCTATCTGTCAC TTCATCAAAA GGACAGTAGA AAAGGAAGGT GGCACCTACA AATGCCATCATTGCGATAAA GGAAAGGCTA TCGTTCAAGA TGCCTCTGCC GACAGTGGTC CCAAAGATGGACCCCCACCC ACGAGGAGCA TCGTGGAAAA AGAAGACGTT CCAACCACGT CTTCAAAGCAAGTGGATTGA TGTGATAACA TGGTGGAGCA CGACACTCTC GTCTACTCCA AGAATATCAAAGATACAGTC TCAGAAGACC AAAGGGCTAT TGAGACTTTT CAACAAAGGG TAATATCGGGAAACCTCCTC GGATTCCATT GCCCAGCTAT CTGTCACTTC ATCAAAAGGA CAGTAGAAAAGGAAGGTGGC ACCTACAAAT GCCATCATTG CGATAAAGGA AAGGCTATCG TTCAAGATGCCTCTGCCGAC AGTGGTCCCA AAGATGGACC CCCACCCACG AGGAGCATCG TGGAAAAAGAAGACGTTCCA ACCACGTCTT CAAAGCAAGT GGATTGATGT GATATCTCCA CTGACGTAAGGGATGACGCA CAATCCCACT ATCCTTCGCA AGACCCTTCC TCTATATAAG GAAGTTCATTTCATTTGGAG AGGACACGCT GAAATCACCA GTCTCTCTCT ACAAATCTAT CTCTCTCGATTCGCGAGCTC GGTACCCGGG gatcgatccSEQ. No. 11: KpnI-lox-BglII-lox-HindIII fragmentggtaccATAACTTCGTATAATGTATGCTATACGAAGTTATagatctATAACTTCGTATAATGTATGCTATACGAAGTTATaagctt

Seq. ID No. 12. Translational coupling of bar and aadA according toscheme in FIG. 18. BglII-SpeI fragment. GAGATCTGgg aggaataact tATGggggtcgacATAACTT CGTATAATGT ATGCTATACG AAGTTATtaG AAGCGGTGAT CGCCGAAGTATCGACTCAAC TATCAGAGGT AGTTGGCGTC ATCGAGCGCC ATCTCGAACC GACGTTGCTGGCCGTACATT TGTACGGCTC CGCAGTGGAT GGCGGCCTGA AGCCACACAG TGATATTGATTTGCTGGTTA CGGTGACCGT AAGGCTTGAT GAAACAACGC GGCGAGCTTT GATCAACGACCTTTTGGAAA CTTCGGCTTC CCCTGGAGAG AGCGAGATTC TCCGCGCTGT AGAAGTCACCATTGTTGTGC ACGACGACAT CATTCCGTGG CGTTATCCAG CTAAGCGCGA ACTGCAATTTGGAGAATGGC AGCGCAATGA CATTCTTGCA GGTATCTTCG AGCCAGCCAC GATCGACATTGATCTGGCTA TCTTGCTGAC AAAAGCAAGA GAACATAGCG TTGCCTTGGT AGGTCCAGCGGCGGAGGAAC TCTTTGATCC GGTTCCTGAA CAGGATCTAT TTGAGGCGCT AAATGAAACCTTAACGCTAT GGAACTCGCC GCCCGACTGG GCTGGCGATG AGCGAAATGT AGTGCTTACGTTGTCCCGCA TTTGGTACAG CGCAGTAACC GGCAAAATCG CGCCGAAGGA TGTCGCTGCCGACTGGGCAA TGGAGCGCCT GCCGGCCCAG TATCAGCCCG TCATACTTGA AGCTAGACAGGCTTATCTTG GACAAGAAGA AGATCGCTTG GCCTCGCGCG CAGATCAGTT GGAAGAATTTGTCCACTACG TGAAAGGCGA GATCACCAAG GTAGTCGGCA AATAAATAAC TTCGTATAATGTATGCTATA CGAAGTTATa ctagt Seq. ID No. 13. CRE-induced expression ofrecombinant protein according to design in FIG. 19. SacI-NheI fragment.gagctcGCTC CCCCGCCGTC GTTCAATGAG AATGGATAAG AGGCTCGTGG GATTGACGTGAGGGGGCAGG GATGGCTATA TTTCTGGGAG AATTAACCGA TCGACGTGCa AGCGGACATTTATTTTaAAT TCGATAATTT TTGCAAAAAC ATTTCGACAT ATTTATTTAT TTTATTATTATGgggATAAC TTCGTATAAT GTATGCTATA CGAAGTTATt aGAAGCGGTG ATCGCCGAAGTATCGACTCA ACTATCAGAG GTAGTTGGCG TCATCGAGCG CCATCTCGAA CCGACGTTGCTGGCCGTACA TTTGTACGGC TCCGCAGTGG ATGGCGGCCT GAAGCCACAC AGTGATATTGATTTGCTGGT TACGGTGACC GTAAGGCTTG ATGAAACAAC GCGGCGAGCT TTGATCAACGACCTTTTGGA AACTTCGGCT TCCCCTGGAG AGAGCGAGAT TCTCCGCGCT GTAGAAGTCACCATTGTTGT GCACGACGAC ATCATTCCGT GGCGTTATCC AGCTAAGCGC GAACTGCAATTTGGAGAATG GCAGCGCAAT GACATTCTTG CAGGTATCTT CGAGCCAGCC ACGATCGACATTGATCTGGC TATCTTGCTG ACAAAAGCAA GAGAACATAG CGTTGCCTTG GTAGGTCCAGCGGCGGAGGA ACTCTTTGAT CCGGTTCCTG AACAGGATCT ATTTGAGGCG CTAAATGAAACCTTAACGCT ATGGAACTCG CCGCCCGACT GGGCTGGCGA TGAGCGAAAT GTAGTGCTTACGTTGTCCCG CATTTGGTAC AGCGCAGTAA CCGGCAAAAT CGCGCCGAAG GATGTCGCTGCCGACTGGGC AATGGAGCGC CTGCCGGCCC AGTATCAGCC CGTCATACTT GAAGCTAGACAGGCTTATCT TGGACAAGAA GAAGATCGCT TGGCCTCGCG CGCAGATCAG TTGGAAGAATTTGTCCACTA CGTGAAAGGC GAGATCACCA AGGTAGTCGG CAAATAAATA ACTTCGTATAATGTATGCTA TACGAAGTTA Ttagctagc

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While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A site specific recombination method for removal of predeterminednucleic acid sequences from the plastid genome, said method comprising:a) providing a first nucleic acid construct, said construct comprising apromoter being operably linked to a nucleic acid encoding plastidtargeting transit sequence which is operably linked to a nucleic acidencoding a site specific recombinase having excision activity, saidconstruct further comprising a first selectable marker encoding nucleicacid having plant specific 5′ and 3′ regulatory nucleic acid sequences;b) providing a second DNA construct, said second construct comprising ansecond selectable marker encoding nucleic acid sequence which is flankedby excision sites, said second construct optionally containing a gene ofinterest, said second construct further comprising flanking plastidtargeting nucleic acid sequences which facilitate homologousrecombination into said plastid genome; c) introducing said second DNAconstruct into a plant cell; d) culturing said plant cell of step c) inthe presence of a selection agent, thereby selecting for those plantcells expressing the proteins encoded by said second DNA construct; e)introducing said first DNA construct into plant cells from step d) inthe presence of a selection agent and selecting those plant cellsexpressing proteins encoded by said first construct, which when presentsaid site specific recombinase acts on said excision sites, therebyremoving said sequence flanked by said excision sites from said plastidgenome.
 2. A method as claimed in claim 1, wherein a plant isregenerated from plant cells of step c), cells are then contacted withsaid first construct and steps d) and e) are performed.
 3. A method asclaimed in claim 1, wherein said first construct is that depicted inFIG.
 3. 4. A method as claimed in claim 1, wherein said second constructis that depicted in FIG.
 2. 5. A method as claimed in claim 1, whereinsaid site specific recombinase is selected from the group consisting ofCRE, flippase, resolvase, FLP, SSV1-encoded integrase, and transposase.6. A method as claimed in claim 1, wherein said excision sites are LOXsequences when CRE recombinase is used and frt sequences when FLPrecombinase is used.
 7. A method as claimed in claim 1, wherein saidselection agent is selected from the group consisting of kanamycin,gentamycin, spectinomycin, streptomycin and hygromycin, phosphinotricin,basta, glyphosate and bromoxynil.
 8. A method as claimed in claim 1,wherein said excision of said predetermined sequence creates anexpressible translational fusion protein.
 9. A method as claimed inclaim 1, wherein said predetermined nucleic acid sequence is theselectable marker encoding nucleic acid present in said secondconstruct.
 10. A plant regenerated from the plant cells of step e) ofclaim
 1. 11. A site specific recombination system comprising theconstructs of the method of claim
 1. 12. A site specific recombinationmethod for removal of predetermined nucleic acid sequences from aplastid genome, said method comprising: a) providing a first nucleicacid construct, said construct comprising a regulated promoter beingoperably linked to a nucleic acid encoding a plastid targeting transitsequence which is operably linked to a nucleic acid encoding a sitespecific recombinase having excision activity, said construct optionallyfurther comprising a first selectable marker encoding nucleic acidhaving plant specific 5′ and 3′ regulatory nucleic acid sequences; b)providing a second DNA construct, said second construct comprising asecond selectable marker encoding nucleic acid, said second constructfurther comprising flanking plastid targeting nucleic acid sequenceswhich flank a pair of excision sites, the second selectable markerencoding nucleic acid, and a predetermined nucleic acid sequence from aplastid genome, wherein the excision sites flank the second selectablemarker encoding nucleic acid and the predetermined nucleic acidsequence, wherein said plant targeting nucleic acid sequences facilitatehomologous recombination into said plastid genome comprising saidpredetermined sequence targeted for deletion; c) introducing said secondDNA construct into a plant cell and allowing said homologousrecombination to occur; d) culturing a plant cell of step c) in thepresence of a selection agent, thereby selecting for those plant cellsexpressing the proteins encoded by said second DNA construct; e)regenerating a plant from cells obtained in step d); f) introducing saidfirst DNA construct into plant cells from the plant of step e) in thepresence of a selection agent and selecting those plant cells expressingproteins encoded by said first construct, which when present said sitespecific recombinase acts on said excision sites, thereby removing saidpredetermined nucleic acid sequence and said second selectable markernucleic acid sequence from the plastid genome.
 13. A method as claimedin claim 12, wherein said regulatable promoter is selected from thegroup of promoters consisting of inducible promoters, tissue specificpromoters, developmentally regulated promoters and chemically induciblepromoters.
 14. A method as claimed in claim 12, wherein saidpredetermined target sequence is selected from the group consisting ofmale sterility genes, clpP ribosomal protein-encoding genes, andribosomal RNA operon sequences.
 15. A method as claimed in claim 12,wherein said protein having excision activity is selected from the groupconsisting of CRE, flippase, resolvase, FLP, SSV1-encoded integrase, andtransposase.
 16. A method as claimed in claim 12, wherein said excisionsites are LOX sequences when CRE recombinase is used, and frt sequenceswhen FLP recombinase is used.
 17. A method as claimed in claim 12,wherein said selection agent is selected from the group consisting ofkanamycin, gentamycin, spectinomycin, streptomycin and hygromycin,phosphinotricin, basta, glyphosate and bromoxynil.
 18. A plantregenerated from the plant cells of step f) of claim
 12. 19. A sitespecific recombination system for removal of predetermined nucleic acidsequences comprising the constructs of claim
 12. 20. Progeny plantsobtained from the plant of claim
 10. 21. Progeny plants obtained fromthe plant of claim 18.